This proposal is focused on the mechanisms through which the tuberous sclerosis complex (TSC) proteins, tuberin and hamartin, repress estrogen-mediated signals. Tuberin (TSC2) is known to interact with estrogen receptor (ER) alpha, and has been previously shown to inhibit ER-driven transcription. We have uncovered an additional, novel pathway linking tuberin and Rheb with 17-beta-estradiol (E2)-mediated signaling. Rheb was recently identified as the target of tuberin's highly conserved GTPase activating domain. Rheb activates the mammalian target of rapamycin (mTOR). In preliminary studies, we found that MCF-7 cells exposedto E2 rapidly convert Rheb to its inactive GDP-bound state. E2exposure also rapidly activates S6 Kinase (S6K), a key target of mTOR. Activation of S6K by E2 is inhibited by tuberin expression. These data indicate that tuberin and Rheb transduce signals from E2 to mTOR. Our central hypothesis is that tuberin inhibits both rapid ("non-genomic") and transcriptional E2- dependent signaling pathways. To address this hypothesis, we propose the following Specific Aims: 1) To determine whether tuberin inhibits estradiol's activation of mTOR and S6K; 2) To determine the mechanism of tuberin's repression of ER-driven transcription; 3) To determine whether estradiol promotes the survival and metastasis of cells lacking tuberin, using both in vitro and in vivo models. This project will define the central estrogen signaling pathways that are regulated by tuberin and Rheb. These pathways may underlie the striking female predominance of lymphangiomyomatosis (LAM). Tuberin and Rheb are expressedin most normal tissues, and the clinical manifestations of TSC impact many organ systems, including the CMS.Estrogen-mediated signals are critical to many normal developmental and metabolic events and estrogen is linked to high prevalence human diseases. We strongly believe, therefore, that the results of this project will have broad medical and biological significance. Lay summary. Estrogen triggers many different cellular events. This project is focused on a protein, tuberin, which appears to inhibit estrogen's actions in the cytoplasm and in the nucleus. The results of this project will elucidate the cause of a female-predominant disease, lymphangiomyomatosis (LAM), and may also contribute to our understanding of other estrogen-linked diseases.